The goal of the proposed research is to develop proteomics technology for drug discovery and medical diagnostics. Specifically we aim to develop a microfluidics-enabled, two-dimensional electrophoresis device that has a potential to replace conventional two-dimensional gel electrophoresis (2DGE). In addition to popular proteomics techniques such as mass spectrometry (MS) and protein microarrays, 2DGE remains one of the most potent methods in proteomics studies. The key advantage of 2DGE is its ability to compare samples with controls for identification of biomarkers of diseases while its major limitations are poor reproducibility and time-consuming processes. To address the limitations, we will develop a 2D electrophoresis device consisting of one channel for the first dimension (isoelectric focusing) and a number of orthogonal channels for the second dimension (polyacrylamide gel electrophoresis). An array of microfluidic pseudovalves will be created for introducing different separation media;a fluidic network allows the transfer of proteins from the first to the second dimension. Faster electrophoresis and higher separation resolution are expected due to higher separation voltage, in an analogy to DNA sequencing that has shifted from slab gel electrophoresis to the capillary array format. In addition, the device will provide enhanced reproducibility of 2D maps due to elimination of gelwarping and improved accuracy in protein identification resulting from the elimination of the diffusion and interaction among proteins in adjacent gel lanes. The specific aims include: (1) designing and fabricating the 2D electrophoresis device consisting of an array of microvalves and the fluidic network;(2) demonstrating the device by protein analysis;and (3) validating the device by analyzing the biomarkers associated with traumatic brain injury (TBI). The significance of the research lies in the fact that the 2D electrophoresis device has the potential to replace 2DGE, which is widely used in biological laboratories. Secondly, if the platform is successfully implemented, it will likely become a rapid, disposable, and quantifiable diagnostic tool to help physicians determine the seriousness of TBI and to guide implementation of appropriate medical treatment. In addition, the platform will likely be useful in searching for the association between biomarkers and a variety of other diseases, and in screening/diagnosing a particular disease using the established biomarkers. Public Health Relevance Statement: The proposed research is to develop proteomics technology for drug discovery and medical diagnostics. Specifically we will develop a microfluidics-enabled electrophoresis device that has a potential to replace classical two-dimensional slab gel electrophoresis. The device will be used for detecting the biomarkers associated with traumatic brain injury.